It has been estimated that 70% of advanced breast cancer patients will face the complication of bone metastases. Three processes are pivotal during bone metastatic growth of breast cancer, namely, tumor cell proliferation, angiogenesis, and osteolysis. During tumor-induced osteolysis, a number of cytokines and growth factors are released from the degraded bone matrix. These factors stimulate further tumor growth, tumor angiogenesis, and tumor-induced osteolysis. New therapies should target all relevant processes to halt this powerful feedback loop. Here, we characterized the new 2-methoxyestradiol analogue ENMD-1198 and showed that it is cytotoxic to tumor cells. Moreover, ENMD-1198 showed both antiangiogenic and vascular disruptive properties and was capable of protecting the bone against tumor-induced osteolysis. We confirmed the in vitro data with a series of in vivo experiments showing the beneficial effects of ENMD-1198 and ENMD-1198–based combination treatments of metastatic breast cancer in bone both on tumor progression and on survival with long-term ENMD-1198 treatment. We confirmed the in vivo relevance of the ENMD-1198 protective effect on bone both with X-ray radiographs and microcomputed tomography. In addition, we combined ENMD-1198 treatment with low-dose metronomic cyclophosphamide and the bisphosphonate risedronic acid, leading to a mild increase in treatment efficacy. Mol Cancer Ther; 10(5); 874–82. ©2011 AACR.

The preference of breast cancer to metastasize to bone followed by growth and bone destruction involves specific tumor–host interactions. It has been estimated that 70% of advanced breast cancer patients will face complications of bone metastases, which includes osteolytic lesions resulting in severe bone pain, fracture, hypercalcemia, and nerve compression (1–3).

Several processes are crucial during bone metastatic growth. These processes include tumor growth and tumor–stroma interactions such as local proangiogenic signaling, local activation of the innate immune system, and local suppression of the adaptive immune system (3–6). Regulatory T (Treg) cells are capable of blocking local immune responses. A local increase in Treg cells is a major factor in the tolerance and immune avoidance of tumors (7). Proangiogenic signaling and the local alterations of the immune system both have a positive feedback on tumor growth.

In addition, breast cancer cells are capable of releasing parathyroid hormone–related protein (PTHrP), which activates receptor activator for NF-κB ligand (RANKL) on stromal cells and osteoblasts. RANK-RANKL signaling not only leads to an increase in osteoclastogenesis but also prolongs the lifetime of osteoclasts, resulting in increased bone resorption. During bone resorption, several growth factors and cytokines are released from the bone matrix. These include TGF-β, insulin-like growth factors, fibroblast growth factor (FGF)-1 and FGF-2, platelet-derived growth factor, and bone morphogenic proteins. The released growth factors stimulate tumor cell proliferation and survival. TGF-β induces the upregulation in VEGF expression by tumor cells, leading to an increase in tumor angiogenesis (8). Moreover, TGF-β stimulates the production and release of PTHrP by the tumor cells, resulting in a self-sustaining feedback loop known as the vicious cycle of bone metastases (9–12).

Because of the complex nature of bone metastasis, new treatments should simultaneously target tumor cell proliferation, angiogenesis, immunologic alterations, and halt the cycle of bone metastases. In this study, we describe the in vitro and in vivo efficacy of a combination therapy targeting these major processes involved in metastatic growth in bone. To do so, we combined low-dose metronomic cyclophosphamide (CTX), 2-methoxyestradiol (2ME2; Panzem) derivate ENMD-1198, and the bisphosphonate risedronic acid.

Previous research indicated that the therapeutic effect of metronomic chemotherapy is mainly due to a reduction in tumor angiogenesis rather than direct cytotoxicity (13, 14). In addition, metronomic CTX selectively depletes Treg cells, resulting in an enhanced tumor immune response (15, 16).

The naturally occurring estrogen metabolite 2ME2 (Fig. 1A) has been shown to have antitumor, antiangiogenic, and antiresorptive effects (17, 18). In vivo therapeutic effects of 2ME2 are moderate because of the rapid inactivation via conjugation (positions 3 and 17) and oxidation (position 17). ENMD-1198 is a 2ME2 derivate with reduced metabolic liabilities (Fig. 1B; ref. 19). It displays antiproliferative effects on tumor cells and neoangiogenesis very similar to 2ME2. ENMD-1198 inhibits proangiogenic signaling through downregulating hypoxia-inducible factor I (HIF)-Iα and STAT3, and it has vascular disruptive effects (20–22). To date, no studies on the treatment of bone metastases or bone-specific effects with ENMD-1198 have been published. ENMD-1198 is currently in clinical trial for the treatment of primary breast cancer (23).

Figure 1.

2ME2 and ENMD-1198. A, structural formula of the estrogen metabolite 2ME2. Rapid inactivation of 2ME2 occurs in vivo via oxidation (at position 17) or conjugation (positions 3 and 17, both underlined). B, the 2ME2 derivate ENMD-1198 has been altered at positions 3 and 17, resulting in slower inactivation and increased plasma levels of the active compound in vivo. Both compounds have very low affinity for the estrogen receptor.

Figure 1.

2ME2 and ENMD-1198. A, structural formula of the estrogen metabolite 2ME2. Rapid inactivation of 2ME2 occurs in vivo via oxidation (at position 17) or conjugation (positions 3 and 17, both underlined). B, the 2ME2 derivate ENMD-1198 has been altered at positions 3 and 17, resulting in slower inactivation and increased plasma levels of the active compound in vivo. Both compounds have very low affinity for the estrogen receptor.

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Bisphosphonate treatment blocks osteoclast function, thereby halting the cycle of bone metastatic growth (24–26). It has been shown in vivo that bisphosphonate treatment mainly prevents the development of new metastases while exhibiting a transient effect on already established metastases (27). Also, bisphosphonates reduce skeletal complications and morbidity in patients with bone metastases (28).

The combination treatment of metronomic CTX, bisphosphonates, and ENMD-1198 targets all of the aforementioned major processes involved in osteolytic bone metastasis of breast cancer, namely, tumor cell proliferation, angiogenesis, local immune suppression, and osteolysis. By targeting the tumor stroma, in addition to the cancerous cells, less drug resistance is to be expected over time. The aim of this study was to assess the efficacy of ENMD-1198 treatment on tumor growth and bone destruction in a murine model for osteolytic breast cancer metastases and to evaluate a possible synergy between ENMD-1198, metronomic CTX, and bisphosphonates.

Animals

Pregnant Swiss albino and female athymic mice (BALB/c nu/nu, 4–6 weeks old) mice were acquired from Charles River Laboratories, housed in individually ventilated cages, and food and water were provided ad libitum. All surgical and analytic procedures were done under isoflurane gas anesthesia. Tumor diameters in the animals were measured routinely. Animals were sacrificed by cervical dislocation at the end of the experimental period or when the tumor diameter exceeded 1 cm in concordance with local guidelines for the use of animals in cancer research. Animal experiments were approved by the local committee for animal health, ethics, and research of Leiden University Medical Center.

Cell lines and culture conditions

The cell lines MDA-231-B/Luc+ (hereafter MDA-BO2), a bone-seeking and luciferase-expressing subclone from the human breast cancer MDA-MB-231 (29, 30) cell line, and RAW264.7, a murine osteoclast precursor cell line (American Type Culture Collection), were cultured in DMEM (Invitrogen) containing 4.5 g glucose/L supplemented with 10% fetal calf serum (FCS; Lonza), 100 units/mL penicillin, 50 μg/mL streptomycin (Invitrogen), and 800 μg/mL geneticin/G418 (Invitrogen).

Both cell lines were grown in a humidified incubator at 37°C and 5% CO2. All cell lines were monthly checked for Mycoplasma infection by PCR. Cells were checked routinely for morphologic changes, and no other authentication tests were done on these cell lines because they were acquired in the laboratory.

In vitro viability assays

Both MDA-BO2 and RAW264.7 were plated in a 96-well plate (Costar) in 100 μL medium at a density of 10,000 cells per well and left over night to adhere. The next day the medium was replaced with medium containing the experimental compounds, 6 wells per condition. After 24, 48, or 144 hours, cell viability was measured using a nonradioactive colorimetric MTS viability assay (Promega Benelux) according to the manufacturer's protocol. Optical absorption was measured at 490 nm with a Versamax absorbance microplate reader (Molecular Devices).

In vitro vascularization models

In vitro angiogenesis was measured as outgrowth of endothelial capillary structures from cultures of 17-day-old fetal mouse metatarsal bone explants, as described previously (31). In short, pregnant Swiss albino mice were sacrificed by cervical dislocation at day 17 of gestation and isolated fetal metatarsals were cultured in α-MEM medium containing 10% FCS (Lonza), penicillin (100 units/mL), and streptomycin (50 μg/mL; Invitrogen) and left for 48 hours to adhere. Thereafter, the feed was replaced with medium containing VEGF (50 ng/mL; rhVEGF-A; Oncogene Science). After 10 days of culture, the explants were fixed and stained with ER-MP12 antibody directed against murine PECAM-1 (CD31; kind gift of Dr. P. Leenen, Erasmus University, Rotterdam, the Netherlands).

Images were obtained using a digital camera, and the area of PECAM-1–positive tubular structures was quantified by image analysis using ImageJ 1.43r (Wayne Rasband, NIH).

In vitro effects on newly established vasculature were examined as described previously (32). In short, isolated 17-day-old fetal metatarsals were left to adhere and cultured for 10 days in the presence of VEGF (50 ng/mL) to stimulate capillary network formation. Thereafter, the feed was replaced by medium containing the test substances. After 24 hours, bone explants were fixed and stained for PECAM-1 for further analysis as described before.

In vitro bone resorption assay

Bone resorption was assessed as described earlier (33). In short, pregnant Swiss albino mice were injected with 30 μCi 45Ca (1 Ci/mmol; PerkinElmer) on day 16 of gestational age. The animals were sacrificed on day 17, and the 45Ca prelabeled fetal metatarsals were isolated and precultured in 1 mL of α-MEM (Invitrogen) supplemented with 0.1% BSA in 6-well plates (Costar) for 24 hours. This allowed 45Ca exchange in the bones with the culture medium. The metatarsals were then cultured in 250 μL of α-MEM, 0.1% BSA in 24-well plates for 10 days in the presence of 100 nmol/L PTHrP (aa 1–34; Bachem) with or without additives (n = 6 per condition). Finally, medium was removed and residual 45Ca was extracted from the bones in 5% trichloroacetic acid for 24 hours. The amount of 45Ca in both the culture media and the decalcification fluid was determined by liquid scintillation with a β-counter (Packard 1600 TR). Resorption was expressed as the percentage of 45Ca from the prelabeled explants that are released in the medium during culture [%45Ca release = 45Ca release into medium (cpm)/total45Ca incorporated in metatarsals (cpm) × 100%].

In vivo treatment of bone metastases

MDA-BO2 cells were injected into the right tibiae as described previously (30). In brief, 2 holes were drilled through the bone cortex of the upper right tibia with a 25-gauge needle (25G 5/8; BD Micro-Fine) and bone marrow was flushed out. Subsequently, 250,000 MDA-BO2 cells/10 μL PBS was injected into the right tibiae of 6-week-old nude mice. Three days after intraosseous inoculation of MDA-BO2 cells, the animals were randomly divided in groups (n = 10).

In a first experiment, 4 groups of mice (n = 10) received either ENMD-1198 (200 mg/kg/d, per oral gavage) or CTX (30 mg/kg/d, through the drinking water as described previously (14)) or a combination of ENMD-1198 and CTX or vehicle control. Treatment started at day 7 after inoculation and was continued throughout the experiment. After 6 weeks, the animals were sacrificed by cervical dislocation and hindlimbs were fixed and kept for ex vivo X-ray analysis.

In a second experiment, 4 groups of mice (n = 10) received ENMD-1198 (200 mg/kg/d, per oral gavage) or ENMD-1198 + CTX (30 mg/kg/d, through the drinking water) or ENMD-1198 + CTX + risedronic acid (Procter & Gamble Pharmaceuticals; 1.6 μmol/kg/d, by subcutaneous injection).

Bioluminescence imaging

The progression of cancer cell growth was monitored weekly by bioluminescent imaging (BLI) with the IVIS100 Imaging System (Caliper Life Sciences).

X-ray radiographic and microcomputed tomographic analysis

At the experimental endpoints, mice were sacrificed and the tumor-bearing hindlimbs were removed and assessed for osteolytic lesions by using a Faxitron 43805 system (Hewlett Packard). X-ray radiographs were scanned and subsequently analyzed using Adobe Photoshop (Adobe Systems).

Microcomputed tomographic (μCT) scans were made using a SkyScan 1076 μCT scanner (SkyScan) using a source voltage set to 40 kV and a source current set to 250 μA, with a step size of 0.9 degrees over a trajectory of 180 degrees. Images were taken with a frame average of 3 to reduce noise and an image pixel size of 9.03 μm. Reconstructions were made using nRecon software (SkyScan), with a beam hardening correction set to 20% and a ring artifact correction set to 5. Volume measurements and visualizations were done by using MeVisLab (MeVis Medical Solutions AG).

Statistics

Results are depicted as mean value ± SEM. Differences between groups were determined by 1-way analysis of variance for multiple comparisons followed by Student's t test for differences between 2 specific groups.

ENMD-1198 reduces MDA-BO2 cell viability in vitro

The direct effect of the proposed treatment on tumor cells was assessed in vitro. To do this, the effect of ENMD-1198 on cell viability was investigated using an MTS colorimetric cell viability assay and with drug concentrations ranging from 0.1 to 3.2 μmol/L. The MTS assay was done after 48 hours of exposure to ENMD-1198, which inhibited MDA-BO2 cancer cell viability with an IC50 of approximately 0.8 μmol/L (Fig. 2A).

Figure 2.

ENMD-1198 treatment reduces viability of MDA-BO2 cells in vitro, whereas low-dose CTX does not. A, ENMD-1198 reduces MDA-BO2 cell viability. Cell viability was measured after 48 hours of ENMD-1198 treatment. B, low doses of 4-HC do not reduce MDA-BO2 cell viability. Cell viability was measured after 24 hours (direct effect) and 144 hours (prolonged metronomic treatment effect) of 4-HC treatment. 4-HC is the main active metabolite of CTX.

Figure 2.

ENMD-1198 treatment reduces viability of MDA-BO2 cells in vitro, whereas low-dose CTX does not. A, ENMD-1198 reduces MDA-BO2 cell viability. Cell viability was measured after 48 hours of ENMD-1198 treatment. B, low doses of 4-HC do not reduce MDA-BO2 cell viability. Cell viability was measured after 24 hours (direct effect) and 144 hours (prolonged metronomic treatment effect) of 4-HC treatment. 4-HC is the main active metabolite of CTX.

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The effect of CTX on cell viability was investigated using an MTS colorimetric cell viability assay and with drug concentrations of the active CTX metabolite 4-hydroperoxycyclophosphamide (4-HC), ranging from 1 nmol/L to 10 μmol/L. The low 4-HC concentrations up to 0.1 μmol/L were similar to concentrations measured in vivo during metronomic CTX treatment (34). Cell viability was measured after both 24 and 144 hours of exposure to 4-HC to assess the effects of direct treatment and prolonged exposure, respectively. Neither short- or long-term effects of 4-HC treatment could be observed using drug concentrations up to 0.1 μmol/L (Fig. 2B).

ENMD-1198 has both an antiangiogenic effect and a vascular disruptive effect in vitro

The vascular effect of ENMD-1198 was assessed in vitro. The antiangiogenic effect of ENMD-1198 treatment was investigated using an in vitro fetal metatarsal outgrowth angiogenesis assay. The fetal metatarsal explants contain a multitude of stromal and vascular/endothelial precursor cells. This cellular complexity closely mimics the in vivo situation during angiogenesis (31). Low concentrations of ENMD-1998 slightly enhanced the vascular outgrowth, suggesting a possible biphasic effect of ENMD-1198 on angiogenesis. However, this enhancement was not statistically significant. Higher doses of ENMD-1198 had profound dose-dependent effects on VEGF-stimulated angiogenesis (Fig. 3A and B).

Figure 3.

ENMD-1198 has both an antiangiogenic effect and a vascular disruptive effect in vitro. A, ENMD-1198 treatment inhibits angiogenesis in a mouse fetal metatarsal explants angiogenesis assay. Metatarsals were cultured for 10 days in the presence of VEGF and ENMD-1198 as indicated. After CD31 staining, the cultures were photographed and the surface area covered by the vascular outgrowth was quantified using ImageJ. Indicated statistical significant differences are in comparison with the VEGF-treated control; *, P < 0.05; ***, P < 0.001. All differences in vascular outgrowth between the 3 ENMD-1198 concentrations are statistically significant. B, representative images of the mouse metatarsal angiogenesis assay showing the metatarsal bone surrounded by vascular outgrowth. C, ENMD-1198 treatment disrupts preexisting vasculature in a mouse fetal metatarsal explants vascular disruption assay. The fetal explants were treated with ENMD-1198 for 24 hours as depicted in the graph after 10 days of culture in the presence of VEGF (50 ng/mL). After CD31 staining, the cultures were photographed and the surface area covered by the vasculature was quantified using ImageJ. Indicated statistical significant differences are in comparison with the untreated control; **, P < 0.01; ***, P < 0.001. D, representative images of the mouse metatarsal vascular disruption assay. Note the remaining fragments of endothelial structures, indicating areas where the vascular network has been disrupted.

Figure 3.

ENMD-1198 has both an antiangiogenic effect and a vascular disruptive effect in vitro. A, ENMD-1198 treatment inhibits angiogenesis in a mouse fetal metatarsal explants angiogenesis assay. Metatarsals were cultured for 10 days in the presence of VEGF and ENMD-1198 as indicated. After CD31 staining, the cultures were photographed and the surface area covered by the vascular outgrowth was quantified using ImageJ. Indicated statistical significant differences are in comparison with the VEGF-treated control; *, P < 0.05; ***, P < 0.001. All differences in vascular outgrowth between the 3 ENMD-1198 concentrations are statistically significant. B, representative images of the mouse metatarsal angiogenesis assay showing the metatarsal bone surrounded by vascular outgrowth. C, ENMD-1198 treatment disrupts preexisting vasculature in a mouse fetal metatarsal explants vascular disruption assay. The fetal explants were treated with ENMD-1198 for 24 hours as depicted in the graph after 10 days of culture in the presence of VEGF (50 ng/mL). After CD31 staining, the cultures were photographed and the surface area covered by the vasculature was quantified using ImageJ. Indicated statistical significant differences are in comparison with the untreated control; **, P < 0.01; ***, P < 0.001. D, representative images of the mouse metatarsal vascular disruption assay. Note the remaining fragments of endothelial structures, indicating areas where the vascular network has been disrupted.

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Next, the effect of ENMD-1198 treatment on previously established vasculature was assessed using metatarsal explants and an optimized vascular disruption protocol as previously described (32). Like the metatarsal angiogenesis assay, this assay contains multiple cell complexity, making it a relevant assay mimicking the in vivo situation. The newly established vascular bed in this assay, like tumor vasculature, is immature and does not contain pericytes. Comparable with the angiogenesis assay, low doses of ENMD-1198 ENMD-1998 slightly enhanced the vascular outgrowth without statistical significance. Higher doses of ENMD-1198 disrupted newly established vasculature with an IC50 of approximately 0.8 μmol/L (Fig. 3C and D).

ENMD-1198 reduces the viability of RAW264.7 osteoclast precursor cells and inhibits PTHrP-stimulated bone resorption

The bone effect of ENMD-1198 was assessed in vitro. The effects of ENMD-1198 and 2ME2 on the viability of RAW264.7 osteoclast precursor cells were investigated using an MTS colorimetric cell viability assay and with drug concentrations ranging from 0.1 to 12.8 μmol/L. The MTS assay was done after 48 hours of ENMD-1198 or 2ME2 exposure. Both ENMD-1198 and 2ME2 reduced RAW264.7 cell viability. With an IC50 of approximately 0.4 μmol/L, ENMD-1198 was 4 times more potent than 2ME2 (IC50 ≈ 1.6 μmol/L; Fig. 4A).

Figure 4.

ENMD-1198 has antiresorptive effects in vitro. A, both 2ME2 and ENMD-1198 reduce the viability of RAW264.7 cells, an osteoclast precursor cell line. ENMD-1198 had an IC50 value of approximately 0.4 μmol/L and was shown to be 4 times more potent than 2ME2 (IC50 ≈ 1.6 μmol/L). Cell viability was measured after 48 hours of 2ME2 or ENMD-1198 treatment. Indicated statistical significant differences are in between 2ME2 and ENMD-1198; *, P < 0.05; ***, P < 0.001. B, ENMD-1198 treatment inhibits bone resorption in a mouse fetal metatarsal explants 45Ca release assay. Radioactive 45Ca-labeled murine fetal metatarsals were cultured under the conditions depicted in the graph. The ratio of the amount of 45Ca in the culture medium and the amount of 45Ca in the bone was measured after 10 days of culture. Indicated statistical significant differences are in comparison with the untreated control, stimulated with PTHrP. Although the 5 μmol/L was not significantly different from the untreated control, it was different from the lower treatment concentrations (P = 0.006) when compared with 0.1 μmol/L; **, P < 0.01; ***, P < 0.001.

Figure 4.

ENMD-1198 has antiresorptive effects in vitro. A, both 2ME2 and ENMD-1198 reduce the viability of RAW264.7 cells, an osteoclast precursor cell line. ENMD-1198 had an IC50 value of approximately 0.4 μmol/L and was shown to be 4 times more potent than 2ME2 (IC50 ≈ 1.6 μmol/L). Cell viability was measured after 48 hours of 2ME2 or ENMD-1198 treatment. Indicated statistical significant differences are in between 2ME2 and ENMD-1198; *, P < 0.05; ***, P < 0.001. B, ENMD-1198 treatment inhibits bone resorption in a mouse fetal metatarsal explants 45Ca release assay. Radioactive 45Ca-labeled murine fetal metatarsals were cultured under the conditions depicted in the graph. The ratio of the amount of 45Ca in the culture medium and the amount of 45Ca in the bone was measured after 10 days of culture. Indicated statistical significant differences are in comparison with the untreated control, stimulated with PTHrP. Although the 5 μmol/L was not significantly different from the untreated control, it was different from the lower treatment concentrations (P = 0.006) when compared with 0.1 μmol/L; **, P < 0.01; ***, P < 0.001.

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After investigating the effects of ENMD-1198 on osteoclast precursor cells, a 45Ca release assay was done to investigate whether ENMD-1198 could effectively block PTHrP-stimulated bone resorption. The assay was conducted using 45Ca-labeled fetal mouse metatarsals in serum-free medium with 0.5% BSA. Bone resorption was stimulated by the addition of 100 nmol/L PTHrP to the culture medium. This culture condition is especially suitable to study effects on PTHrP-induced bone resorption (33). ENMD-1198 effectively blocks PTHrP stimulated 45Ca release in a dose-dependent manner (Fig. 4B).

ENMD-1198–based combination treatments inhibit tumor growth in vivo

Following the in vitro characterization of ENMD-1198 on the 3 major processes involved in skeletal metastatic growth of breast cancer, the ENMD-1198–based combination treatment effects were assessed in vivo. To investigate the effect on tumor burden, mice were treated with ENMD-1198, CTX, or ENMD-1198 + CTX. Tumor growth was followed using BLI. In all treated groups, the tumor burden was decreased significantly compared with the untreated controls by day 27. There was a trend that ENMD-1198 treatment was more effective than CTX alone. A difference between ENMD-1198 and the ENMD-1198 + CTX combination could not be observed (Fig. 5A).

Figure 5.

ENMD-1198–based combination treatments reduce tumor burden but does not eradicate the tumor. A, treatment started 7 days after inoculation (T0). All treated groups had a reduced tumor burden compared with the untreated control; *, P < 0.05. There was a trend that the ENMD-1198–treated group had a lower tumor burden than the CTX-treated group (P = 0.063). B, the ENMD-1198 + CTX combination group had a statistically significant decreased tumor burden compared with the control group at day 21 (P = 0.026). There was a trend that the ENMD-1198 and the ENMD-1198 + CTX + risedronic acid–treated groups had a reduced tumor burden compared with the untreated controls (P = 0.088 and P = 0.086, respectively). However, the control mice died before this difference could reach significance. C, survival curves of mice treated with ENMD-1198–based combinations. Mice were inoculated intraosseous with MDA-BO2 cells. Treatment started 7 days after inoculation (T0). Mice were sacrificed when the tumor exceeded 1-cm diameter in accordance with local guidelines. Treatment was stopped after 10 weeks. One mouse in the ENMD-1198–treated group died in week 4 because of an accident with the oral gavage. The tumor size reached a steady state in the treated groups during the treatment period but rapidly expanded after the treatment was stopped. D–F, individual graphs for each of the combination treatments. ENMD-1198 versus control (D). ENMD-1198 + CTX versus control (E). ENMD-1198 + CTX + risedronic acid versus control (F); BP, bisphosphonate (risedronic acid).

Figure 5.

ENMD-1198–based combination treatments reduce tumor burden but does not eradicate the tumor. A, treatment started 7 days after inoculation (T0). All treated groups had a reduced tumor burden compared with the untreated control; *, P < 0.05. There was a trend that the ENMD-1198–treated group had a lower tumor burden than the CTX-treated group (P = 0.063). B, the ENMD-1198 + CTX combination group had a statistically significant decreased tumor burden compared with the control group at day 21 (P = 0.026). There was a trend that the ENMD-1198 and the ENMD-1198 + CTX + risedronic acid–treated groups had a reduced tumor burden compared with the untreated controls (P = 0.088 and P = 0.086, respectively). However, the control mice died before this difference could reach significance. C, survival curves of mice treated with ENMD-1198–based combinations. Mice were inoculated intraosseous with MDA-BO2 cells. Treatment started 7 days after inoculation (T0). Mice were sacrificed when the tumor exceeded 1-cm diameter in accordance with local guidelines. Treatment was stopped after 10 weeks. One mouse in the ENMD-1198–treated group died in week 4 because of an accident with the oral gavage. The tumor size reached a steady state in the treated groups during the treatment period but rapidly expanded after the treatment was stopped. D–F, individual graphs for each of the combination treatments. ENMD-1198 versus control (D). ENMD-1198 + CTX versus control (E). ENMD-1198 + CTX + risedronic acid versus control (F); BP, bisphosphonate (risedronic acid).

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Next we wanted to investigate whether adding a bisphosphonate (risedronic acid) to the ENMD-1198 + CTX combination would improve the treatment outcome. The ENMD-1198 + CTX-treated group had a significantly reduced tumor burden compared with the control by day 21. There was a trend that the ENMD-1198 and the ENMD-1198 + CTX + risedronic acid–treated groups had a reduced tumor burden as well. The animals in the control group were sacrificed before these differences could reach significance because the tumor size in the control group exceeded 1 cm in diameter, a defined endpoint stated in the Dutch guidelines for animal experiments in cancer research. However, differences in growth kinetics between the treated groups and the untreated group can still be appreciated. The tumor development in this experiment was more rapid than in the previous experiment. In conclusion, adding risedronic acid to the treatment did not improve treatment outcome (Fig. 5B).

ENMD-1198–based combination treatments are capable of controlling tumor growth but are unable to eradicate the tumor

After investigating the treatment effects on tumor burden, the efficacy of long-term treatment with ENMD-1198 or ENMD-1198-based combinations was assessed. Seven days after intraosseous inoculation with MDA-BO2 cells, mice were treated with ENMD-1198, ENMD-1198 + CTX, ENMD-1198 + risedronic acid, ENMD-1198 + CTX + risedronic acid, or vehicle control. The treatment continued for a maximum of 10 weeks. In accordance to the Dutch guidelines for animal experiments in cancer research, mice were sacrificed when the tumor diameter exceeded 1 cm. All 3 ENMD-1198–based combination treatments could control tumor growth over a prolonged period of time. The animals receiving the combination of ENMD-1198 and risedronic acid seemed less healthy with a thinner skin and aberrant behavior, suggesting possible adverse effects of long-term treatment with this combination of drugs. The tumors grew exponentially as soon as the treatment was stopped after 10 weeks. The addition of low-dose CTX and/or risedronic acid did not improve treatment outcome (Fig. 5C–F).

ENMD-1198 protects the bone against tumor-induced osteolysis in vivo

Finally, the effect of ENDM-1198 treatment on tumor-induced osteolysis was assessed using both X-ray radiographs and high-resolution μCT scans done ex vivo after sacrificing the mice. These include untreated controls and groups treated with ENMD-1198 or CTX or a combination of ENMD-1198 + CTX. The corresponding BLI data of these animals are depicted in Fig. 5A. In the X-ray radiographs, the surface area of osteolytic lesions was measured after manually drawing a region of interest around the lesion. The osteolytic lesions were significantly smaller in groups that received ENMD-1198 than the groups that did not receive ENMD-1198 (Fig. 6A).

Figure 6.

ENMD-1198 treatment protects the bone against tumor-induced osteolysis in vivo. A, quantification of osteolytic lesion size by surface measurement on X-ray radiographs, which were obtained after termination of the experiment. The lesion surface was measured as the number of pixels. Graphs show the average and SEM. Groups treated with ENMD-1198 have smaller lesions than the other groups; **, P < 0.01; ***, P < 0.001. B, quantification of osteolytic lesion size volume measurement in μCT scans, which were made after termination of the experiment. Bone volumes of the upper half of the tibia (region of interest is indicated in the inset), cutoff planes were always defined in a 90-degree angle to the bone. Graphs show the average and SEM. Groups treated with ENMD-1198 had a larger bone volume than the other groups, confirming the radiograph data; *, P < 0.05; **, P < 0.01. C, representative X-ray and μCT images of all the treatment groups. Both X-ray and μCT analyses show a reduction in osteolysis in ENMD-1198–treated groups.

Figure 6.

ENMD-1198 treatment protects the bone against tumor-induced osteolysis in vivo. A, quantification of osteolytic lesion size by surface measurement on X-ray radiographs, which were obtained after termination of the experiment. The lesion surface was measured as the number of pixels. Graphs show the average and SEM. Groups treated with ENMD-1198 have smaller lesions than the other groups; **, P < 0.01; ***, P < 0.001. B, quantification of osteolytic lesion size volume measurement in μCT scans, which were made after termination of the experiment. Bone volumes of the upper half of the tibia (region of interest is indicated in the inset), cutoff planes were always defined in a 90-degree angle to the bone. Graphs show the average and SEM. Groups treated with ENMD-1198 had a larger bone volume than the other groups, confirming the radiograph data; *, P < 0.05; **, P < 0.01. C, representative X-ray and μCT images of all the treatment groups. Both X-ray and μCT analyses show a reduction in osteolysis in ENMD-1198–treated groups.

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Because the radiograph method is based on a manual definition of the osteolytic lesion, we wanted to confirm these results in using a method that is less susceptible to bias. To do so, the bone volumes of the upper half of the tibiae of these mice were measured in μCT scans. The groups that received ENMD-1198 had a significantly larger bone volume than the groups that did not receive ENMD-1198, confirming the X-ray radiographic data (Fig. 6B).

This study was the first to assess the treatment efficacy of the 2ME2-derived compound ENMD-1198 on osteolytic bone metastasis of breast cancer. Our results indicate that ENMD-1198 interferes with all the crucial processes of bone metastatic growth of breast cancer, namely, tumor cell proliferation, angiogenesis, and bone resorption. We confirmed our in vitro data with a series of in vivo experiments showing the beneficial effects of ENMD-1198 and ENMD-1198–based combination treatments of metastatic breast cancer in bone both on tumor progression and on survival with long-term treatment over a period of 10 weeks. Furthermore, we showed the in vivo relevance of the ENMD-1198 protective effect on bone.

The observed in vitro effects of ENMD-1198 on cell viability were in agreement with earlier findings (19). ENMD-1198 and 2ME2 are microtubule-disrupting agents. Similar to other microtubule-disrupting agents, they interfere with cell mitosis and thus have profound antiproliferative effects on tumor cells and during neoangiogenesis whereas resting cells are unaffected. Moreover, 2ME2 causes the phosphorylation of Bcl-2 and Bcl-xL, leading to an upregulation of the intrinsic and extrinsic apoptotic pathways, a mechanism shared with paclitaxel (35).

ENMD-1198 and 2ME2 reduce angiogenesis by inhibiting the expression and nuclear accumulation of HIF-Iα. This results in a downregulation of VEGF expression and local proangiogenic signaling (21, 22, 36, 37). This impaired hypoxia sensing increases local concentrations of radical oxygen species, enhancing the proapoptotic effects of these compounds (21, 22). In addition, ENMD-1198 interferes with endothelial cell motility, chemotaxis, and morphogenesis into capillary-like structures (22). The results of our in vitro angiogenesis assay and vascular disruption assay confirmed these findings.

Metronomic CTX inhibits angiogenesis via 3 different mechanisms: by direct induction of apoptosis of proliferating tumor endothelial cells, by blocking or reducing the viability of circulating endothelial progenitor cells, and by elevating levels of cellular and circulating thrombospondin-1 (TSP-1). These antiangiogenic effects are enhanced in combination with VEGF inhibitors (13, 14). Our finding that in vitro treatment with 4-HC is not cytotoxic to breast cancer cells were in agreement with earlier experiments conducted by Bocci and colleagues (13).

We expected to find a strong synergy between the antiangiogenic effects of ENMD-1198 and metronomic CTX treatments in vivo because both treatments have been reported to inhibit angiogenesis via different mechanisms. The combination of CTX + ENMD-1198 resulted in a better treatment outcome than CTX treatment alone, but this difference was not profound enough to suggest any synergy between CTX and ENMD-1198. Moreover, adding CTX to ENMD-1198 treatment did improve the overall outcome compared with ENMD-1198 alone in one in vivo experiment but did not in the other.

ENMD-1198 reduced osteoclast precursor cell viability and could inhibit PTHrP-stimulated bone resorption in vitro. When comparing the CTX-treated group and the ENMD-1198–treated groups, there was a significant 60% reduction in osteolytic lesion size in the ENMD-1198–treated groups. The differences in tumor burden were much smaller and not significant between all groups. CTX treatment did not improve the osteolytic phenotype compared with the control group, even though the tumor burden was significantly lower. Taken together, we conclude a strong bone specific effect of ENMD-1198 treatment.

To date, this is the first study showing these bone-specific effects of ENMD-1198. It has previously been shown that 2ME2 inhibits osteoclast differentiation and bone resorption and is cytotoxic to osteoclasts (38). Interestingly, a 2ME2-mediated upregulation of osteoprotegerin has been described in osteosarcoma cells. This induction of osteoprotegerin can contribute to the antiresorptive functions of 2ME2 in osteolytic metastatic lesions (39, 40). Induction of osteoprotegerin might be part of the mechanism in which ENMD-1198 protects the bone matrix from degeneration. The addition of risedronic acid to the combination treatment did not impact tumor growth, nor did it prolong the survival of the animals.

In conclusion, ENMD-1198 treatment of osteolytic bone metastases has profound in vivo effects on both tumor growth and osteolysis. ENMD-1198–based combination treatments can control tumor growth; however, the tumor continue to grow after treatment is stopped, indicating a strong cytostatic effect. Because of the potent cytostatic and antiangiogenic effects of ENMD-1198, only a weak additional effect of having CTX treatment could be observed. However, any additional value of CTX treatment might be stronger in an immunocompetent model because of a selective depletion of Treg cells (41). ENMD-1198 has strong antiresorptive properties and the addition of risedronic acid to the treatment did not improve the overall treatment outcome. ENMD-1198 is a promising new compound in the field of metastatic bone disease affecting all processes that are crucial during development and growth of bone metastases.

No potential conflicts of interest were disclosed.

ENMD-1198 was a generous gift from Dr. Tony Treston (Entremed Inc.). The authors thank Dr. Alan Chan for proofreading the manuscript.

This work has been supported by the Dutch Cancer Society Koningin Wilhelmina Fonds (UL2007-3801; T.J.A. Snoeks) and the sixth FP EU grants EMIL (LSHC-CT-2004-503569; I.M. Mol, C.W.G.M. Löwik) and DiMI (LSBH-CT-2005-512146; I. Que, E.L. Kaijzel, C.W.G.M. Löwik).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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